Int. J. Dev. Biol. 55: 85 - 92 (2011)

https://doi.org/10.1387/ijdb.103119ev

Vol 55, Issue 1

Development of a fast, objective, quantitative methodology to monitor angiogenesis in the chicken chorioallantoic membrane during development

Technical Article | Published: 21 February 2011

Eva Verhoelst*^,1,2, Bart De Ketelaere¹, Veerle Bruggeman², Eduardo Villamor³, Eddy Decuypere² and Josse De Baerdemaeker¹

¹Faculty of Bioscience Engineering, Department of Biosystems, Division of MeBioS, K.U.Leuven, Belgium, ²Faculty of Bioscience Engineering, Department of Biosystems, Division of Livestock-Nutrition-Quality, K.U.Leuven, Belgium and 3Department of Pediatrics, Maastricht University Medical Center (MUMC+), GROW School for Oncology and Developmental Biology, Maastricht, The Netherlands

Abstract

Recent research investigates the role of different gas concentrations during incubation, on chicken growth, quality and health post hatch. One of the parameters of chicken development which changes under different gas concentrations is angiogenesis in the chorioallantoic membrane (CAM). To be able to perform large incubation experiments under different conditions, angiogenesis in the whole CAM must be quantified objectively and easily. In this paper, a fast, objective, quantitative methodology to assess changes in the overall vascular development in the CAM of chicken embryos is presented. Samples were taken with minimal disturbance by emptying the egg, so that the CAM stayed attached to the shell, which was then cut in pieces. We employed a commercial digital camera and a macro lens set at 5x magnification to take pictures with sufficient contrast and resolution (2.64 mm/pixel). These were processed with computer algorithms to calculate the vascular fraction (VF) and the fractal dimension (FD) automatically on binary images. The ratio of the repeatability and reproducibility variation compared to the parts variation was 0.32 for VF and 0.21 for FD. In a validation experiment (n=284), one group was incubated under hypoxic conditions and the other under normoxic conditions. It was shown that early hypoxia stimulated angiogenesis, while chronic hypoxia impeded growth with significant differences between both groups, which is in accordance with literature data. Thus, we report here a method to asses overall angiogenesis in the CAM under different incubation conditions.

Keywords

angiogenesis, chicken embryo, CAM, hypoxia, image analysis